It is becoming evident that methylation of the genome is an example of epigenetics observed in a wide range of biological species from Escherichia coli to plants to vertebrates, and this is associated with various living phenomena. Particularly in mammals, the methylation is also becoming an important area of study in view of ontogenesis, cell differentiation, canceration, and the like, and methylation of CpG islands in the promoter region of a gene is known to inactivate tumor-suppressor genes.
The methylation of a genome specifically arises from the methylation of cytosine contained in nucleic acid. The bisulfite method is currently most widely used as a method for detecting the methylated state of the cytosine contained in nucleic acid is a method utilizing the fact that bisulfite treatment of nucleic acid as a specimen does not convert methylcytosine and converts only cytosine to uracil (Patent Literatures 1 and 2). When after bisulfite treatment, the resultant is subjected to PCR and sequenced, uracil is detected as thymine and methylcytosine is detected as cytosine. The presence or position of methylation can be determined from the difference between cytosine and thymine (uracil) produced before and after treatment. However, the disadvantages of the bisulfite method are that its sequencing operation is cumbersome, the method has a long reaction time (typically a dozen hours or so) for complete modification, and the treatment often produces a depurination reaction and often causes fragmentation of the sample; thus, the method requires improvement.
As a method other than this method, a quantitation method for methylcytosine using an anti-methylcytosine antibody has been reported (Patent Literature 3). However, the quantitation method for methylcytosine using the antibody can measure the total amount of methylcytosine contained in a nucleic acid of interest, but does not enable it to be determined at which position in the base sequence cytosine is methylated. For gene expression, it is crucial to know which cytosines are methylated, not just the frequency of cytosine methylation.
The present inventors have recently reported a method for selectively quantitating the methylation of cytosine at a specific position in a base sequence using an anti-methylcytosine antibody together with experimental results using synthetic oligomers (Patent Literature 4). This method involves placing the cytosine to be detected at a specific position in a nucleic acid in a DNA bulge formed by mismatching when the nucleic acid hybridizes with a complementary strand and measuring the amount of an anti-cytosine antibody or an anti-methylcytosine antibody binding to the nucleic acid; this enables it to be determined whether or not the cytosine to be detected is methylated.